Haemophagocytic lymphohistiocytosis after intravesical BCG administration for bladder cancer presenting with multiorgan failure

Bacillus Calmette-Guérin (BCG), is administered intravesically as an adjuvant immunotherapy for the treatment of non-muscle invasive bladder cancer. While mild non-infectious problems can occur in up to 85 % of cases, significant local and systemic complications have been reported in 1–5 % of cases. We report the case of a patient with superficial bladder cancer who developed multiorgan failure after intravesical BCG instillation including the kidney and liver with subsequent haemophagocytic lymphohistiocytosis. Our case illustrates the first reported combination of secondary haemophagocytic lymphohistiocytosis with severe renal and liver failure after BCG immunotherapy for bladder carcinoma. Treatment strategy is discussed.


INTRODUCTION
Bacillus Calmette-Guérin (BCG), is a live attenuated strain of Mycobacterium bovis that is administered intravesically as an adjuvant immunotherapy for the treatment of non-muscle invasive bladder cancer [1].It is usually well tolerated and minor non-infectious side-effects such as fever, malaise, and bladder irritation (dysuria or moderate hematuria) occurring a few hours after intravesical BCG treatment are fairly common (up to 85%) [2], self-limiting (resolving within 48 h) and indicative of a local hypersensitivity reaction [3].However, significant local and systemic complications can occur in a small percentage of individuals (1-5 %) [2,3].Clinical symptoms might present anywhere between hours and years after BCG treatment.The treatment approach is empirical based mainly on the reported experience from case series.Therefore, clinicians may be challenged with more severe systemic complications that occur when BCG organisms gain access to the systemic circulation most likely due to uroepithelial disruption [4].
We report the case of a patient with superficial bladder cancer who developed severe multi-organ failure (MOF) and haemophagocytic lymphohistiocytosis after intravesical BCG instillation.

CASE PRESENTATION
A 73-year-old man with a history of hypertension and dyslipidaemia presented with painless, grossly visible hematuria eighteen months before admission.Cystoscopy identified three papillary tumours that were treated with transurethral resection (TUR).Histology revealed a T1 tumour, and imaging investigations ruled out any metastases (Stage 1).Surveillance cystoscopy 6 months later revealed a recurrence of stage one bladder cancer which was followed by second TUR.A month later, weekly BCG induction immunotherapy for 6 weeks was started.However, the third instillation was traumatic, followed by self-limiting dysuria, and the patient refused to complete induction treatment.Five months before admission, he opted to continue with maintenance 3 weekly sessions every 3 months.The first course was unremarkable, but almost a month before admission, and a few hours after the second instillation of the second maintenance course (the eighth instillation in total), he complained of fever, fatigue, and hematuria.His symptoms were initially attributed to bladder infection, and he was treated with amikacin and ciprofloxacin (for three and ten days respectively) without symptom remission.
He was then referred to a district hospital where blood tests revealed acute renal (cr=4.64mg dl −1 ) and liver failure (ALT=65 U/l, INR=2.0,TBil=21.41 mg dl −1 , ALP=379 U/l, g-gt=642 U/l) requiring two courses of haemodialysis.Methylprednisolone 80 mg/day and a triple anti-tuberculosis treatment (ATT) with isoniazid, rifampicin, and ethambutol was initiated, only to be discontinued 3 days later due to significant biochemical and clinical deterioration (creatinine=5.36,TBIL=25.66).He was then transferred to our hospital twenty days after his last BCG instillation.On admission, patient was lethargic and jaundiced while his labs showed stage three acute kidney injury (AKI), marked hyperbilirubinemia with moderate cholestasis, and pancytopenia (Table 1).His peripheral blood smear showed marked anisopoikilocytosis, toxic granulation, stacking of erythrocytes with basophilic stippling, and erythroblasts (~8 % of nucleated erythrocytes in different maturation stages).An abdominal ultrasound with Doppler imaging revealed only hepatosplenomegaly while a chest CT showed bibasilar alveolar consolidations with air bronchograms and bilateral effusions.
He was started on broad spectrum antibiotic coverage with meropenem, daptomycin and tetracycline.Further laboratory workup revealed a markedly increased ferritin, hypertriglyceridemia and hypofibrinaemia.A bone marrow (BM) aspiration and biopsy were performed showing active haemophagocytosis of erythroblasts from BM stromal cells (Fig. 1a, b).Bone marrow histology revealed well-formed non-caseating granulomas with several histiocytes and a Langerhans giant cell (Fig. 1c, d).BM Ziehl-Nielsen staining and cultures in Lowenstein-Jensen medium as well as Mycobacteria Growth Indicator Tube (MGIT) for Mycobacteria were all negative.
The above laboratory findings established the diagnosis of secondary haemophagocytic lymphohistiocytosis (HLHscore=337, prob-ability=99.99%) and on the second hospital day, the patient was started on high dose methylprednisolone (2 mg/kg/day) and intravenous immunoglobulin (IVIG, 0.4 gm/kg/day for 5 days).By day six his clinical status as well as his laboratory values gradually improved while his blood, urine cultures remained negative and antibiotics were discontinued.On day 13, an increase in the aminotransferase levels was noted (Table 1) probably attributed to rapid steroid tapering, and the patient was given three daily pulses of intravenous methylprednisolone (0.5 gm) with gradual improvement.A second BM aspiration was then performed and an XPERT MTB/RIF ultra assay for detection of Mycobacterium tuberculosis complex DNA returned positive (without mutations in the rpoB genefor rifampicin resistance).On day 20, his previous triple anti-tuberculous regimen was gradually reinitiated without any adverse events (considering his G6PD deficiency and recent renal and liver failure).patient was discharged home on day 33 in good health on rifampicin, isoniazid, ethambutol, pyridoxine, calcium, and Vitamin D3 supplementation with a gradual taper of his methylprednisolone dose.Throughout his follow-up, his anti-TB therapy was well tolerated (Table 1).

DISCUSSION
Secondary HLH due to BCG intravesical administration is rare, with only a few cases reported in the literature [5][6][7][8][9].According to those and the present case it affects males between the ages of 49 and 78, and it manifests as constitutional symptoms hours or days after the last BCG instillation.The patient's age is comparable with the mean age of bladder cancer patients treated with BCG immunotherapy [2], as well as those complicated with BCGitis [3].Furthermore, HLH caused by intravesical BCG administration might occur between the first and sixth BCG instillation [5][6][7][8][9], although in our case it appeared immediately after the eighth instillation.In the largest review [3], the median number of instillations before systemic BCGitis developed was 6.0 (Q1-Q3 range: 4.0-9.0).The reason for the wide range of instillations preceding HLH or BCGitis incidence is unknown.The multi-organ systemic manifestations following BCG administration could be the result of either a systemic hypersensitivity reaction to M. bovis (as evidenced by noncaseating granulomas in the liver [8] and kidneys [10] in the absence of viable mycobacteria in the respective tissues) or a direct result of a disseminated M. bovis infection (as evidenced by viable organisms in different organs and/or positive molecular tests).However, a combination of these two mechanisms may be implicated in some cases, resulting in significant macrophage activation and secondary HLH, as was most likely the case with our patient.BM (6/6 of patients), liver (5/6), kidneys (3/6), and lungs (2/6) or their combination are the most often affected organs in HLH caused by BCG instillation.Diagnosis is mostly based upon clinical suspicion taking into account patient's history of recent BCG administration in association with histological findings of granulomas and/or microbiological detection of M. bovis in affected organs.
Treatment consists of high-dose glucocorticoids (methylprednisolone 2-2.5 mg/kg/day or dexamethasone 10 mg/m 2 ) combined with IVIG (0.4 g/kg/day for 5 days) and triple anti-TB therapy (rifampicin, isoniazid and ethambutol for 2 months followed by rifampicin and isoniazid for another 7 months since M. bovis present in BCG is considered resistant to pyrazinamide) [3].However, it should be noted that early initiation of triple ATT and especially, before the implementation of IVIG and steroids, in cases with liver or renal involvement, may result in early discontinuation or a switch to an alternative ATT (4/6 of the cases) until biochemistry abnormalities resolve.Of notice, even though mortality associated with HLH secondary to tuberculosis is very high 45-50 % [11], all reported patients with BCG-associated HLH recovered.
The possibility that HLH was not caused by BCG and that something else developed in the patient led to a severe clinical scenario of BCG infection with severe dysregulation due to HLH is remote, as BCG was detected in the patient's BM and extensive laboratory work-up ruled out other potential aetiologies for secondary HLH.Infectious (including HIV, Hepatitis viruses, Parvovirus B19, CMV, EBV, Leishmania, Brucella, Leptospira, Rickettsia spp., and syphilis serology), rheumatologic (ANA, ANCA autoantibodies, ENA screening, serum complement and immunoglobulin levels) or malignant (full body imaging and negative serum cancer markers) diseases were clinically and laboratory ruled out.The patient had no history of past tuberculosis infection and had never received immunosuppressive therapy.This is the first case of secondary HLH caused by disseminated M. bovis infection following intravesical BCG treatment, with severe MOF (stage 3 AKI necessitating haemodyalisis and liver failure indicated by encephalopathy, INR prolongation, and severe hyperbilirubinemia).In a similar reported case [7] organ manifestations were much milder.The mechanisms involved in BCG-associated HLH with MOF, whether infectious or hypersensitivity forms of BCGitis, are various features of the same illness, and BCG-related sequelae appear to be a continuum, with active infection at one end and hypersensitivity reaction at the other.In any case, prompt diagnosis and early initiation of the appropriate immunomodulatory and anti-mycobacterial treatment is crucial for patient's survival.
-"The before the last paragraph of Discussion needs to be rephrased.Still discussing the multiorgan failure, but without making emphasis that this is the first report of it." Response: the last paragraph of the Discussion section was rephrased (lines 165-168) and the first report of multiorgan failure associated with HLH after intravesical BCG case is being emphasized.
-"Some aspects still need to be discussed, such as the age of the patient and the fact that this didn't occur in the previous 8 instillations with BCG." Response: a new text concerning patient's age compatibility with other reported HLH cases, with the mean age of patients with bladder cancer treated with BCG immunotherapy, and those presenting BCGitis, was added in the first paragraph of the Discussion section (lines 124-128).In addition, a new phrase referring to the number of instillations that preceded both HLH secondary to BCGitis and the systemic complications of BCG administration in general was added (lines 128-133).

Response to Reviewer #2 -"in the manuscript page 4 line 138 kindly correct the word manifestation to manifestations"
Response: the correction mentioned above was done (line 168).
-"in the Discussion page 4, line 139: Is the number (11) a reference number?If so, please write it in the same format as other references" Response: indeed number ( 11) is a reference number and the citation was corrected in the same format as the other references.

VERSION 1
Editor recommendation and comments https://doi.org/10.1099/acmi.0.000670.v1.5 © 2023 de Dios R.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.

Comments:
The manuscript has been reviewed by two experts in the field, who have raised minor concerns that would need to be fixed prior to publication.This includes suggestions of text structure rearrangements and additional aspects to discuss, which will need to be specifically amended.Additionally, the title needs to be amended by using a standard letter casing.The manuscript is well-written; however, I have two comments: In the discussion, page 4, line 136: Kindly correct the word manifestation to manifestations.In the discussion, page 4, line 139: Is the number (11) a reference number?If so, please write it in the same format as other references.

Please rate the quality of the presentation and structure of the manuscript Good
To extent are the conclusions supported by the data?Strongly support

Is there a potential financial or other conflict of interest between yourself and the author(s)? No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes Reviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000670.v1.3 © 2023 Cervantes J.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.
Jorge Cervantes; Nova Southeastern University -Fort Lauderdale/Davie Campus: Nova Southeastern University, UNITED STATES https://orcid.org/0000-0002-4359-5951Date report received: 16 August 2023 Recommendation: Minor Amendment Comments: 1. Description of the case(s) Good, may benefit from stating the bladder cancer diagnosis first, and then progressing to the BCG instillations, and why these were initiated.2. Presentation of results This is a case report.BM images are excellent.3. How the style and organization of the paper communicates and represents key findings Reorganize content in the Introduction and the Discussion 4. Literature analysis or discussion Satisfactory.5. Any other relevant comments The case report is indeed interesting.Furthermore, the successful recovery of the patient merits highlight of the authors management.The abstract seems to contain more introductory information than the Introduction itself.Suggest moving the first part of the Discussion to the Introduction.Discussion should be to discuss their findings.Was HLH due to BCG, or something that developed in the patient, leading to a severe clinical scenario of BCG infection in a patient with a severe immunedysregulation due to the HLH?The before the last paragraph of the Discussion need to be rephrased.Still discussing the multiorgan failure, but without making emphasis that this is the first report of it.Some aspects still need to be discussed, such as the age of the patient and the fact that this didn't occur in the previous 8 instillations with BCG.

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data?Strongly support

Is there a potential financial or other conflict of interest between yourself and the author(s)? No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes SciScore report https://doi.org/10.1099/acmi.0.000670.v1.1 © 2023 The Authors.This is an open-access article report distributed under the terms of the Creative Commons License.
Dios; Brunel University London, Life Sciences, UNITED KINGDOM Date report received: 06 September 2023 Recommendation: Minor Amendment

Reviewer 2
recommendation and comments https://doi.org/10.1099/acmi.0.000670.v1.4 © 2023 AbdulRahman E. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Eiman AbdulRahman; Cairo University, Clinical pathology, 6-October city, 24 B Makka Al-Mokarama street, Cairo, Egypt Date report received: 04 September 2023 Recommendation: Minor Amendment Comments: 1. Description of the case: Satisfactory.2. Presentation of results: NA. 3. How the style and organization of the paper communicates and represents key findings: Good. 4. Literature analysis or discussion: Good. 5. Any other relevant comments:

Table 1 .
Laboratory findings at admission, during hospitalization and follow-